This summer I worked in the Kurre Lab at the Children’s Hospital of Philadelphia to investigate the potential of tissue-based cyclic immunofluorescence (t-CyCIF) on Fanconi Anemia (FA) research. FA is a recessive autosomal disease caused by mutations in FA genes. FA patients typically show phenotypic abnormalities including deformed fingers and short stature and face early onset bone marrow failure. A prior study by Yoon et al. indicates that during development, specifically at E12.5, Fancd2 deficient murine samples exhibit a fetal liver mass and a hematopoietic stem cell deficit. Given such findings, my project was directed at in utero murine fetal livers to identify key distinctions in microenvironment between wildtype and Fancd2 knockout samples.
To image the samples, we utilized the tissue-based cyclic (t-CyCIF) immunofluorescence technique which follows a cyclic procedure including nuclear staining, 4-channel imaging, bleaching, and antibody incubation which is then proceeded by a washing step and repeated. As opposed to traditional imaging methods, t-CyCIF allows for more than four antibodies to be investigated per sample while utilizing conventional laboratory instruments and reagents.
This technique was completed using CD-150, CD-31, and Dlk-1 antibodies which stain for stem, endothelial, and stromal cells, respectively. We hypothesized that there is a fundamental difference in architecture and microenvironment between wildtype and Fancd2 knockout murine fetal liver. Specifically, we expect there to be fewer stem, endothelial, and stromal cells in the knockout Fancd2 murine fetal liver which will be exhibited through a lower frequency of fluorescence signal for CD-150, CD-31, and Dlk-1 markers.
This summer research experience has equipped me with a myriad of technical laboratory skills vital for biomedical research ranging from software expertise to gel electrophoresis. In addition, through networking with faculty and exploring different avenues within medicine such as dual degree programs and medical specialties, my initial interest in medicine has only strengthened from this internship. I plan to further develop the skills I have obtained by applying them to both my pre-med courses and other research positions.